DNA polymerase

ABSTRACT

The present invention relates to a DNA polymerase possesses the properties of 1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, as compared to the case where an activated DNA is used as a substrate; 2) possessing a 3′→5′ exonuclease activity; 3) being capable of amplifying a DNA fragment of about 20 kbp, in the case where polymerase chain reaction (PCR) is carried out using λ-DNA as a template. It also relates to a DNA polymerase-constituting protein; a DNA containing the base sequence encoding thereof; and a method for producing the DNA polymerase. The present invention provides a novel DNA polymerase possessing both a high primer extensibility and a 3′→5′ exonuclease activity.

This application is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/JP96/03869 which has an Internationalfiling date of Dec. 26, 1996 which designated the United States ofAmerica, the entire contents of which are hereby incorporated byreference.

TECHNICAL FIELD

The present invention relates a DNA polymerase which is useful as areagent for genetic engineering, a method for producing the same, and agene encoding thereof.

BACKGROUND ART

DNA polymerases are useful enzymes as reagents for genetic engineering,and the DNA polymerases are widely used for method of determining basesequences of DNA, labeling, methods of site-directed mutagenesis, andthe like. Also, thermostable DNA polymerases have recently been remarkedwith the development of the polymerase chain reaction (PCR) method, andvarious DNA polymerases suitable for the PCR method have been developedand commercialized.

Presently known DNA polymerases can be roughly classified into fourfamilies according to amino acid sequence homologies, among which familyA (pol I type enzymes) and family B (α type enzymes) account for thegreat majority. Although DNA polymerases belonging to each familygenerally possess mutually similar biochemical properties, detailedcomparison reveals that individual DNA polymerase enzymes differ fromeach other in terms of substrate specificity, substrate analogincorporation, degree and rate for primer extension, mode of DNAsynthesis, association of exonuclease activity, optimum reactionconditions of temperature, pH and the like, and sensitivity toinhibitors. Thus, those possessing especially suitable properties forthe respective experimental procedures have been selectively used of allavailable DNA polymerases.

DISCLOSURE OF INVENTION

An object of the present invention is to provide a novel DNA polymerasenot belonging to any of the above families, and possessing biochemicalproperties not owned by any of the existing DNA polymerases. Forexample, primer extension activity and 3′→5′ exonuclease activity areconsidered as mutually opposite properties, and none of the existing DNApolymerase enzymes with strong primer extension activity possess 3′→5′exonuclease activity, which is an important proofreading function forDNA synthesis accuracy. Also, the existing DNA polymerases possessingexcellent proofreading functions are poor in primer extension activity.Therefore, development of a DNA polymerase possessing both potent primerextension activity and potent 3′→5′ exonuclease activity wouldsignificantly contribute to DNA synthesis in vitro.

Another object of the present invention is to provide a method forproducing the novel DNA polymerase mentioned above.

A still another object of the present invention is to provide a geneencoding the DNA polymerase of the present invention.

As a result of extensive investigation, the present inventors have foundgenes of the novel DNA polymerase from hyperthermophilic arcaebacteriumPyrrococcus furious, followed by cloning of the above genes, and haveclarified that two kinds of novel proteins possess a novel DNApolymerase activity exhibiting the activity under coexistence of theabove two kinds of proteins. Furthermore, the present inventors haveprepared a transformant into which the above genes are introduced, andhave succeeded in mass-producing the complex type DNA polymerase.

Accordingly, the gist of the present invention is as follows:

[1] A DNA polymerase characterized in that the DNA polymerase possessesthe following properties:

1) exhibiting higher polymerase activity when assayed by using as asubstrate a complex resulting from primer annealing to a single strandedtemplate DNA, as compared to the case where an activated DNA is used asa substrate;

2) possessing a 3′→5′ exonuclease activity;

3) being capable of amplifying a DNA fragment of about 20 kbp, in thecase where polymerase chain reaction (PCR) is carried out using λ-DNA asa template under the following conditions:

PCR Conditions:

(a) a composition of reaction mixture: containing 10 mM Tris-HCl (pH9.2), 3.5 mM MgCl₂, 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP,0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10pmole/50 μl primer λ1 (SEQ ID NO:8 in Sequence Listing), primer λ11 (SEQID NO:9 in Sequence Listing), and 3.7 units/50 μl DNA polymerase;

(b) reaction conditions: carrying out a 30-cycle PCR, wherein one cycleis defined as at 98° C. for 10 seconds and at 68° C. for 10 minutes;

[2] The DNA polymerase according to the above item [1], characterized inthat the DNA polymerase exhibits a lower error rate in DNA synthesis ascompared to Taq DNA polymerase;

[3] The DNA polymerase according to the above item [1] or [2], whereinthe molecular weight as determined by gel filtration method is about 220kDa or about 385 kDa;

[4] The DNA polymerase according to any one of the above items [1] to[3], characterized in that the DNA polymerase exhibits an activity undercoexistence of two kinds of DNA polymerase-constituting protein, a firstDNA polymerase-constituting protein and a second DNApolymerase-constituting protein;

[5] The DNA polymerase according to the above item [4], characterized inthat the molecular weights of the first DNA polymerase-constitutingprotein and the second DNA polymerase-constituting protein are about90,000 Da and about 140,000 Da as determined by SDS-PAGE, respectively;

[6] The DNA polymerase according to the above item [4] or [5],characterized in that the first DNA polymerase-constituting proteinwhich constitutes the DNA polymerase according to the above item [4] or[5] comprises the amino acid sequence as shown by SEQ ID NO:2 inSequence Listing, or is a functional equivalent thereof possessingsubstantially the same activity which results from deletion, insertion,addition or substitution of one or more amino acids in the amino acidsequence;

[7] The DNA polymerase according to the above item [4] or [5],characterized in that the second DNA polymerase-constituting proteinwhich constitutes the DNA polymerase according to the above item [4] or[5] comprises the amino acid sequence as shown by SEQ ID NO:4 inSequence Listing, or is a functional equivalent thereof possessingsubstantially the same activity which results from deletion, insertion,addition or substitution of one or more amino acids in the amino acidsequence;

[8] The DNA polymerase according to item [4] or [5], characterized inthat the first DNA polymerase-constituting protein which constitutes theDNA polymerase according to the above item [4] or [5] comprises theamino acid sequence as shown by SEQ ID NO:2 in Sequence Listing, or is afunctional equivalent thereof possessing substantially the same activitywhich results from deletion, insertion, addition or substitution of oneor more amino acids in the amino acid sequence, and that the second DNApolymerase-constituting protein which constitutes the DNA polymeraseaccording to the above item [4] or [5] comprises the amino acid sequenceas shown by SEQ ID NO:4 in Sequence Listing, or is a functionalequivalent thereof possessing substantially the same activity whichresults from deletion, insertion, addition or substitution of one ormore amino acids in the amino acid sequence;

[9] A first DNA polymerase-constituting protein which constitutes theDNA polymerase according to the above item [4] or [5], wherein the firstDNA polymerase-constituting protein comprises the amino acid sequence asshown by SEQ ID NO:2, or an amino acid sequence resulting from deletion,insertion, addition or substitution of one or more amino acids in theamino acid sequence, as a functional equivalent thereof possessingsubstantially the same activity;

[10] A second DNA polymerase-constituting protein which constitutes theDNA polymerase according to the above [4] or [5], wherein the second DNApolymerase-constituting protein comprises the amino acid sequence asshown by SEQ ID NO:4, or an amino acid sequence resulting from deletion,insertion, addition or substitution of one or more amino acids in theamino acid sequence as a functional equivalent thereof possessingsubstantially the same activity;

[11] A DNA containing a base sequence encoding the first DNApolymerase-constituting protein according to the above item [9],characterized in that the DNA comprises an entire sequence of a basesequence encoding the amino acid sequence as shown by SEQ ID NO:2 inSequence Listing, or a partial sequence thereof, or that the DNA encodesa protein having an amino acid sequence resulting from deletion,insertion, addition or substitution of one or more amino acids in theamino acid sequence of SEQ ID NO:2 and possessing a function as thefirst DNA polymerase-constituting protein;

[12] A DNA containing a base sequence encoding the first DNApolymerase-constituting protein according to the above items [9],characterized in that the DNA comprises an entire sequence of the basesequence as shown by SEQ ID NO:1 in Sequence Listing or a partialsequence thereof, or that the DNA comprises a base sequence capable ofhybridizing thereto under stringent conditions;

[13] A DNA containing a base sequence encoding the second DNApolymerase-constituting protein according to the above item [10],characterized in that the DNA comprises an entire sequence of a basesequence encoding the amino acid sequence as shown by SEQ ID NO:4, or apartial sequence thereof, or that the DNA encodes a protein having anamino acid sequence resulting from deletion, insertion, addition orsubstitution of one or more amino acids in the amino acid sequence ofSEQ ID NO:4 and possessing a function as the second DNApolymerase-constituting protein;

[14] A DNA containing a base sequence encoding the second DNApolymerase-constituting protein according to the item [10],characterized in that the DNA comprises an entire sequence of the basesequence as shown by SEQ ID NO:4 in Sequence Listing or a partialsequence thereof, or that the DNA comprises a base sequence capable ofhybridizing thereto under stringent conditions;

[15] A method for producing a DNA polymerase, characterized in that themethod comprises culturing a transformant containing both gene encodingthe first DNA polymerase-constituting protein according to the aboveitem [11] or [12], and gene encoding the second DNApolymerase-constituting protein according to the above item [13] or[14], and collecting the DNA polymerase from the resulting culture; and

[16] A method for producing a DNA polymerase, characterized in that themethod comprises culturing a transformant containing gene encoding thefirst DNA polymerase-constituting protein according to the above item[11] or [12], and a transformant containing gene encoding the second DNApolymerase-constituting protein according to the above item [13] or[14], separately; mixing DNA polymerase-constituting proteins containedin the resulting culture; and collecting the DNA polymerase.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a restriction endonuclease map of the DNA fragment insertedinto the cosmid Clone No. 264 and the cosmid Clone No. 491 obtained inExample 1.

FIG. 2 shows a restriction endonuclease map of an XbaI-XbaI DNA fragmentinserted into a plasmid pFU1001.

FIG. 3 is a graph for an optimum pH of the DNA polymerase of the presentinvention.

FIG. 4 is a graph for a heat stability of the DNA polymerase of thepresent invention.

FIG. 5 is a graph for a 3′→5′ exonuclease activity of the DNA polymeraseof the present invention.

FIG. 6 is an autoradiogram for a primer extension activity of the DNApolymerase of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

(1) DNA Polymerase of Present Invention and Constituting ProteinsThereof

An example of the DNA polymerase of the present invention has thefollowing properties:

1) exhibiting higher polymerase activity when assayed by using as asubstrate a complex resulting from primer annealing to a single strandedtemplate DNA, as compared to the case where an activated DNA (DNaseI-treated calf thymus DNA) is used as a substrate;

2) possessing a 3′→5′ exonuclease activity;

3) optimum pH being between 6.5 and 7.0 (in potassium phosphate buffer,at 75° C.);

4) exhibiting a remaining activity of about 80% after heat treatment at80° C. for 30 minutes;

5) being capable of amplifying a DNA fragment of about 20 kbp, in thecase where polymerase chain reaction (PCR) is carried out using λ-DNA asa template under the following conditions:

PCR conditions:

(a) composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2),3.5 mM MgCl₂, 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01%bovine serum albumin (BSA), 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10pmole/50 μl primer λ1 (SEQ ID NO:0:8 in Sequence Listing), primer λ11(SEQ ID NO:9 in Sequence Listing), and 3.7 units/50 μl DNA polymerase.Here, one unit of the DNA polymerase is defined as follows: Fiftymicroliters of a reaction mixture [20 mM Tris-HCl (pH 7.7), 15 mM MgCl₂,2 mM 2-mercaptoethanol, 0.2 mg/ml activated DNA, 40 μM each of dATP,dCTP, dGTP and dTTP, 60 nM [³H]-dTTP (manufactured by Amersham)],containing a sample to assay activity, is reacted at 75° C. for 15minutes. A 40 μl portion of this reaction mixture is spotted onto a DEpaper (manufactured by Whatman) and washed with 5% Na₂HPO₄ five times.Thereafter, the remaining radioactivity on the DE paper is measuredusing a liquid scintillation counter, and the amount of the enzymecausing the incorporation of 10 nmol of [³H]-dTMP per 30 minutes into asubstrate DNA is defined as one unit of the enzyme; and

(b) PCR conditions: carrying out a 30-cycle PCR, wherein one cycle isdefined as at 98° C. for 10 seconds and at 68° C. for 10 minutes; and

6) The DNA polymerase of the present invention is superior to the TaqDNA polymerase in terms of both primer extension activity and accuracyof DNA synthesis. Specifically, the DNA polymerase of the presentinvention is superior to the Taq DNA polymerase, a typical thermostableDNA polymerase (e.g., TaKaRa Taq, manufactured by Takara Shuzo Co.,Ltd.), in terms of primer extension properties in DNA synthesisreaction, for instance, DNA strand length capable of DNA amplificationby PCR method, and accuracy of DNA synthesis reaction (low error rate inDNA synthesis).

The DNA polymerase of the present invention is an enzyme constituted bytwo kinds of proteins, wherein a molecular weight of the DNA polymeraseof the present invention is about 220 kDa or about 385 kDa, asdetermined by gel filtration, and also shown by two bands correspondingto about 90,000 Da and about 140,000 Da on SDS-PAGE, respectively. Theprotein of about 90,000 Da (corresponding to ORF3 as described below) isherein referred to as the first DNA polymerase-constituting protein, andthe protein of about 140,000 Da (corresponding to ORF4 as describedbelow) is herein referred to as the second DNA polymerase-constitutingprotein. It is assumed that in the DNA polymerase of the presentinvention, the first DNA polymerase-constituting protein and the secondDNA polymerase-constituting protein are non-covalently bonded to form acomplex in a molar ratio of 1:1 or 1:2.

The first DNA polymerase-constituting protein which constitutes the DNApolymerase of the present invention may comprise the amino acid sequenceshown by SEQ ID NO:2 in Sequence Listing, or may be a functionalequivalent possessing substantially the same activity. Also, the secondDNA polymerase-constituting protein may comprise the amino acid sequenceshown by SEQ ID NO:4 in Sequence Listing, or may be a functionalequivalent possessing substantially the same activity.

The term “a functional equivalent” as described in the presentspecification is defined as follows. A protein existing in nature canundergo mutation, such as deletion, insertion, addition andsubstitution, of amino acids in an amino acid sequence thereof owing tomodification reaction and the like of the protein itself in vivo orduring purification, besides causation such as polymorphism and mutationof the genes encoding it. However, it has been known that there are someproteins which exhibit substantially the same physiological activitiesor biological activities as a protein without mutation. Those proteinshaving structural differences as described above without recognizing anysignificant differences of the functions and the activities thereof, arereferred to as “a functional equivalent.” Here, the number of mutatedamino acids is not particularly limited, as long as the resultingprotein exhibits substantially the same physiological activities orbiological activities as a protein without mutation. Examples thereofinclude one or more of mutations, for instance, one or severalmutations, more specifically one to about ten mutations (such asdeletion, insertion, addition and substitution) and the like.

The same can be said for the resulting proteins in the case where theabove mutation is artificially introduced into the amino acid sequenceof a protein. In this case, more diverse mutants can be prepared. Forexample, although the methionine residue at the N-terminus of a proteinexpressed in Escherichia coli is reportedly often removed by the actionof methionine aminopeptidase, since the methionine residue is notremoved perfectly depending on the kinds of proteins, those havingmethionine residue and those without methionine residue can be bothproduced. However, the presence or absence of the methionine residuedoes not affect protein activity in most cases. It is also known that apolypeptide resulting from substitution of a particular cysteine residuewith serine in the amino acid sequence of human interleukin 2 (IL-2)retains IL-2 activity [Science, 224, 1431 (1984)].

In addition, during the production of a protein by genetic engineering,the desired protein is often expressed as a fusion protein. For example,purification of the desired protein is facilitated by adding theN-terminal peptide chain derived from another protein to the N-terminusof the desired protein to increase the amount of expression of thedesired protein, or by adding an appropriate peptide chain to the N- orC-terminus of the desired protein, expressing the protein, and using acarrier having affinity for the peptide chain added. Accordingly, a DNApolymerase having an amino acid sequence which has a partial differencewith that of the DNA polymerase of the present invention is within thescope of the present invention as “a functional equivalent,” as long asit exhibits substantially the same activities as the DNA polymerase ofthe present invention.

(2) Gene of DNA Polymerase of Present Invention

The DNA encoding the first DNA polymerase-constituting protein whichconstitutes the DNA polymerase of the present invention includes a DNAcomprising an entire sequence of the base sequence encoding the aminoacid sequence as shown by SEQ ID NO:2 in Sequence Listing or a partialsequence thereof including, for instance, a DNA comprising an entiresequence of the base sequence as shown by SEQ ID NO:1 or a partialsequence thereof. Specifically, a DNA comprising a partial sequence ofthe base sequence encoding the amino acid sequence as shown by SEQ IDNO:1 including, for instance, the DNA comprising a partial sequence ofthe base sequence as shown by SEQ ID NO:1 in Sequence Listing, the basesequence encoding a protein possessing a function of the first DNApolymerase-constituting protein is also included in the scope of thepresent invention. Also, in the amino acid sequence as shown by SEQ IDNO:2, the above DNA also includes a DNA encoding a protein comprising anamino acid sequence resulting from deletion, insertion, addition,substitution and the like of one or several amino acids, the proteinpossessing a function of the first DNA polymerase-constituting protein.Furthermore, a base sequence capable of hybridizing to the above basesequences under the stringent conditions, the base sequence encoding aprotein possessing a function of the first DNA polymerase-constitutingprotein, is also included in the scope of the present invention. Inaddition, the DNA encoding the second DNA polymerase-constitutingprotein which constitutes the DNA polymerase of the present inventionincludes a DNA comprising an entire sequence of the base sequenceencoding the amino acid sequence as shown by SEQ ID NO:3 in SequenceListing or a partial sequence thereof including, for instance, a DNAcomprising an entire sequence of the base sequence as shown by SEQ IDNO:3 in Sequence Listing or a partial sequence thereof. Specifically,the DNA comprising a partial sequence of the base sequence encoding theamino acid sequence as shown by SEQ ID NO:4, for instance, the DNAcomprising a partial sequence of the base sequence as shown by SEQ IDNO:3 in Sequence Listing, the base sequence encoding a proteinpossessing a function of the second DNA polymerase-constituting protein,is also included in the scope of the present invention. Also, in theamino acid sequence as shown by SEQ ID NO:4, the above DNA also includesa DNA encoding a protein comprising an amino acid sequence resultingfrom deletion, insertion, addition, substitution and the like of one orseveral amino acids, the protein possessing a function of the second DNApolymerase-constituting protein. Furthermore, a base sequence capable ofhybridizing to the above base sequences under the stringent conditions,the base sequence encoding a protein possessing a function of the secondDNA polymerase-constituting protein, is also included in the scope ofthe present invention.

The term “protein possessing a function of the first DNApolymerase-constituting protein” or “protein possessing a function ofthe second DNA polymerase-constituting protein” herein refers to aprotein possessing properties exhibiting a DNA polymerase activity withvarious physicochemical properties shown in the above items 1) to 6).

Here, the term “capable of hybridizing under the stringent conditions”refer to hybridizing to a probe, after incubating at 50° C. for 12 to 20hours in 6×SSC (wherein 1×SSC shows 0.15 M NaCl, 0.015 M sodium citrate,pH 7.0) containing 0.5% SDS, 0.1% bovine serum albumin (BSA), 0.1%polyvinyl pyrrolidone, 0.1% Ficol 400, and 0.01% denatured salmon spermDNA with the probe.

The term “DNA containing a base sequence encoding an amino acidsequence” described in the present specification will be explained. Oneto six kinds are known to exist for each amino acid with regards to acodon (triplet base combination) for designating a particular amino acidon the gene. Therefore, there can be a large number of DNA encoding anamino acid sequence, though depending on the amino acid sequence. Innature, genes do not always exist in stable forms, and it is not rarefor genes to undergo mutations on a base sequence. There may be a casewhere mutations on the base sequence do not give rise to any changes inan amino acid sequence to be encoded (referred to as silent mutation).In this case, it can be said that different kinds of genes encoding thesame amino acid sequence have been generated. The possibility,therefore, cannot be negated for producing a variety of genes encodingthe same amino acid sequence after many generations of the organism evenwhen a gene encoding a particular amino acid sequence is isolated.

Moreover, it is not difficult to artificially produce a variety of genesencoding the same amino acid sequence by means of various geneticengineering techniques. For example, when a codon used in the naturalgene encoding the desired protein is used at a low frequency in the hostin the production of the protein by genetic engineering, the amount of aprotein expressed is sometimes low. In this case, high expression of thedesired protein is achieved by artificially converting the codon intoanother one used at a high frequency in the host without changing theamino acid sequence encoded (for instance, Japanese Patent Laid-Open No.Hei 7-102146). As described above, it is, of course, possible toartificially produce a variety of genes encoding a particular amino acidsequence. Such artificially produced different polynucleotides are,therefore, included in the scope of the present invention, as long asthe gene encodes the amino acid sequence disclosed in the presentinvention.

(3) Method for Producing DNA Polymerase of Present Invention

The present inventors have found genes of a novel DNA polymerase from ahyperthermophilic archaebacterium, Pyrococcus furiosus, and cloned toclarify that the genes encode a novel DNA polymerase exhibiting itsactivity by the coexistence of two kinds of proteins on the genes. Inthe present invention, the DNA polymerase of the present invention canbe mass-produced by preparing transformants incorporating the abovegenes. For this purpose, the transformant may be prepared by a processcomprising culturing a transformant containing both the gene encodingthe first DNA polymerase-constituting protein and the gene encoding thesecond DNA polymerase-constituting protein, and collecting the DNApolymerase from the resulting culture. Alternatively, the transformantmay be prepared by a process comprising separately culturing atransformant containing the gene encoding the first DNApolymerase-constituting protein and a transformant containing the geneencoding the second DNA polymerase-constituting protein, mixing the DNApolymerase-constituting proteins contained in the resulting culture, andcollecting the DNA polymerase therefrom.

Here, the phrase “transformant containing both the gene encoding thefirst DNA polymerase-constituting protein and the gene encoding thesecond DNA polymerase-constituting protein” may be a transformantresulting from co-transformation with two expression vectors containingthe respective genes, or it may be a transformant prepared byrecombining both genes into one expression vector to allow therespective proteins to be expressed.

(4) A Cloning of the Genes of the DNA Polymerase of the PresentInvention, an Analysis of Obtained Clones, Physicochemical Properties,Activities, Applicabilities to PCR Method of Expression Product DNAPolymerase, and the Like are Hereinafter Described in Detail.

The strain used for the present invention is not subject to particularlimitation. Examples thereof include Pyrococcus furiousus DSM3638, as astrain belonging to the genus Pyrococcus. The above strain can be madeavailable from Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH. In the case of culturing the above strain in an appropriate growthculture, preparing a crude extract from the resulting culture, andsubjecting the crude extract to a polyacrylamide gel electrophoresis,since the present inventors found existences of several kinds of proteinbands showing DNA polymerase activity in the gel, it has beenanticipated that the genes corresponding to these respective bands haveexisted. Specifically, the novel DNA polymerase gene and the productthereof can be cloned by the procedures exemplified below.

1) DNA is extracted from Pyrococcus furiousus;

2) The DNA obtained in 1) is digested with an appropriate restrictionendonuclease, to prepare a DNA library with a plasmid, cosmid and thelike, as a vector;

3) The library prepared in 2) is introduced into Escherichia coli, and aforeign gene is expressed to prepare a protein library in which crudeextracts of the resulting clones are collected;

4) A DNA polymerase activity is assayed by using the protein libraryprepared in 3), and a foreign DNA is taken out from the Escherichia coliclone which provides a crude extract having an activity;

5) The Pyrococcus furiousus DNA fragment contained in the plasmid orcosmid taken out is analyzed to narrow down the gene region encoding aprotein exhibiting a DNA polymerase activity;

6) The base sequence of the region in which the protein exhibiting a DNApolymerase activity is presumably encoded is determined to deduce theprimary structure of the protein; and

7) An expression plasmid is constructed to take a form which more easilyallows the expression of the protein deduced in 6) in Escherichia coli,and the produced protein is purified and analyzed for the propertiesthereof.

The above DNA donor, Pyrococcus furiousus DSM3638, is ahyperthermophilic archaebacterium, which is cultured at 95° C. underanaerobic conditions. Known methods can be used as a method fordisrupting grown cells followed by extracting and purifying DNA, amethod for digesting the obtained DNA with a restriction endonucleaseand for other methods. Such methods are described in detail by inMolecular Cloning: A Laboratory Manual, 75-178, published by Cold SpringHarbor Laboratory in 1982, edited by T. Maniatis et al.

In the preparation of a DNA library, the triple helix cosmid vector(manufactured by Stratagene), for example, can be used. The DNA ofPyrococcus furiousus is partially digested with Sau3AI (manufactured byTakara Shuzo Co., Ltd.), and the digested DNA is subjected to densitygradient centrifugation to obtain the long DNA fragments. They areligated to the BamHI site of the above vector, followed by in vitropackaging. The respective transformants obtained from the DNA librarythus prepared are separately cultured. After harvesting, cells aredisrupted by ultrasonication, and the resulting disruption isheat-treated to inactivate the DNA polymerase from the host Escherichiacoli. Thereafter, a supernatant containing a thermostable protein can beobtained by centrifugation. The above supernatant is named as a cosmidprotein library. By means of assaying the DNA polymerase activity usinga portion of the supernatant, a clone that expresses the DNA polymerasederived from Pyrococcus furiousus can be obtained. DNA polymeraseactivity can be assayed using the known method described in DNAPolymerase from Escherichia coli, published by Harpar and Row, edited byD. R. Davis, 263-276 (authored by C. C. Richardson).

One of the DNA polymerase genes of Pyrococcus furiosus has already beencloned and its structure clarified by the present inventors, asdescribed in Nucleic Acids Research, 21, 259-265 (1993). The translationproduct of the above gene is a polypeptide having a molecular weight ofabout 90,000 Da and consisting of 775 amino acids, and the amino acidsequence thereof clearly contains preserved sequences of the α-type DNApolymerases. In fact, since the DNA polymerase activity exhibited bythis gene product is inhibited by aphidicolin, which is a specificinhibitor of α-type DNA polymerases, the above DNA polymerase isdistinguishable from the DNA polymerase of the present invention.Therefore, the above known gene out of the obtained clones exhibitingthermostable DNA polymerase activity can be removed by a processcomprising digesting the cosmid contained in each clone, carrying outhybridization with the above gene as a probe, and selecting anunhybridizing clone. A restriction endonuclease map of the DNA insertcan be prepared for the cosmid digested with the resulting clonecontaining the novel DNA polymerase gene. Next, a location of the DNApolymerase gene on the above DNA fragment can be determined by a processcomprising dividing the above DNA fragment into various regions on thebasis of the obtained restriction endonuclease map, subcloning eachregion into a plasmid vector, introducing the resulting vector intoEscherichia coli, and assaying the thermostable DNA polymerase activityexhibited therein. An XbaI-XbaI DNA fragment of about 10 kbp containingthe DNA polymerase gene can be thus obtained.

The recombinant Escherichia coli harboring a plasmid incorporating theabove DNA fragment exhibits a sufficient level of a DNA synthesisactivity in the crude extract thereof even after treatment at 90° C. for20 minutes, while such an activity is not found in any plasmids withoutincorporating a DNA fragment. Therefore, it can be concluded that theinformation for producing a thermostable polymerase is present on theDNA fragment, and that a gene having the above information is expressedin the above Escherichia coli. The plasmid resulting from recombinationof the DNA fragment into a pTV118N vector (manufactured by Takara ShuzoCo., Ltd.) is named as pFU1001. The Escherichia coli JM109 transformedwith the above plasmid is named and identified as Escherichia coliJM109/pFUFU1001, has been deposited under accession number FERM BP-5579with the National Institute of Bioscience and Human-Technology, Agencyof Industrial Science and Technology, Ministry of International Tradeand Industry, of which the address is 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, 305, Japan, since Aug. 11, 1995 (date of original deposit)under the Budapest Treaty.

The base sequence of the DNA fragment inserted in the plasmid pFUFU1001can be determined by a conventional method, for instance, by the dideoxymethod. Furthermore, regions capable of encoding a protein in the basesequence, i.e., open reading frames (ORFs), can be deduced by analyzingthe resulting base sequence.

An 8,450 bp sequence in the base sequence of the XbaI-XbaI DNA fragmentof about 10 kbp inserted in the plasmid pFU1001 is shown by SEQ ID NO:5in Sequence Listing. In the base sequence, there are six consecutiveORFs, named as ORF1, ORF2, ORF3, ORF4, ORF5, and ORF6, respectively,naming from the 5′ terminal side. FIG. 2 shows the restrictionendonuclease map of the above XbaI-XbaI fragment and the location of theORFs on the fragment (ORF1 to ORF6, from the left in the Figure).

A sequence showing homologies to any of known DNA polymerases was notfound in any one of the above six ORFs. It should be noted, however,that on ORF1 and ORF2, there is a sequence homologous to the CDC6protein found in Saccharomyces cerivisiae, or a sequence homologous tothe CDC18 protein found in Schizosaccharomyces pombe. The CDC6 and theCDC18 are anticipated as proteins that are necessary for the cell cycleshift to the DNA synthesis phase (S phase) in yeasts, the proteinsregulating initiation of the DNA replication. Also, the ORF6 has asequence homologous to the RAD51 protein, known to act in DNA damagerepair in yeasts and recombination in the somatic mitosis phase and inthe meiosis phase in yeasts, and a sequence homologous to the Dmclprotein, a meiosis phase-specific homolog to the RAD51 protein. The geneencoding the RAD51 protein is also known to be expressed at the cellcycle shift from the G1 to S phase. For the other ORFs, namely ORF3,ORF4, and ORF5, there have been no known proteins found to have ahomologous sequence.

It is possible to determine from which of the above ORFs thethermostable DNA polymerase activity is derived by a process comprisingpreparing recombinant plasmids inserted with the respective DNAfragments deleting various regions, transforming a host with theplasmids, and assaying the thermostable polymerase activity of eachtransformant obtained. The transformant resulting from transformationwith a recombinant plasmid inserted with a DNA fragment prepared bydeleting ORF1 or ORF2, or deleting ORF5 or ORF6, from the aboveXbaI-XbaI DNA fragment of about 10 kbp retains the thermostable DNApolymerase activity, while those resulting from transformation with arecombinant plasmid inserted with a DNA fragment prepared by deletingORF3 or ORF4 loses its activity. This fact predicts that the DNApolymerase activity is encoded by ORF3 or ORF4.

It is possible to determine by which of ORF3 and ORF4 the DNA polymeraseis encoded by a process comprising preparing recombinant plasmidsseparately inserted with the respective ORFs, transforming a host witheach recombinant plasmid, and assaying exhibition of a thermostable DNApolymerase activity in each transformant obtained. Unexpectedly, onlyvery weak DNA polymerase activity is detected in a crude extractobtained from the transformant containing ORF3 or ORF4 alone. However,since a similar level of a thermostable DNA polymerase activity to thatin the transformant containing both ORF3 and ORF4 can be obtained in thecase where the two extracts are mixed, it is shown that the novel DNApolymerase of the present invention requires the actions of thetranslation products of the two ORFs. It is possible to find out whetherthe two proteins form a complex to exhibit the DNA polymerase activity,or one modifies the other to convert it to an active enzyme bydetermining the molecular weight of the DNA polymerase. The results ofthe determination of the molecular weight of the above DNA polymerase bygel filtration method demonstrate that the above two proteins form acomplex.

The base sequence of ORF3 is shown by SEQ ID NO:1 in Sequence Listing,and the amino acid sequence of the ORF3-derived translation product,namely the first DNA polymerase-constituting protein as deduced from thebase sequence, is shown by SEQ ID NO:2. The base sequence of ORF4 isshown by SEQ ID NO:3 in Sequence Listing, and the amino acid sequence ofthe ORF4-derived translation product, namely the second DNApolymerase-constituting protein as deduced from the base sequence, isshown by SEQ ID NO:4.

The DNA polymerase of the present invention can be expressed in cells byculturing a transformant resulting from transformation with arecombinant plasmid into which both ORF3 and ORF4 are introduced, forinstance, Escherichia coli JM109/pFUFU1001, under usual culturingconditions, for instance, culturing in an LB medium (10 g/l trypton, 5g/l yeast extract, 5 g/l NaCl, pH 7.2) containing 100 μg/ml ampicillin.The above polymerase can be purified from the above cultured cells tothe extent that only the two kinds of bands of nearly two kinds of theDNA polymerase-constituting proteins are obtained in SDS-polyacrylamidegel electrophoresis (SDS-PAGE), by carrying out ultrasonication, heattreatment, and chromatography using an anionic exchange column (RESOURCEQ column, manufactured by Pharmacia), a heparin Sepharose column (HiTrapHeparin, manufactured by Pharmacia), a gel filtration column (Superose6HR, manufactured by Pharmacia) or the like. It is also possible toobtain the desired DNA polymerase by a process comprising separatelyculturing transformants respectively containing ORF3 or ORF4 alone asdescribed above, and subsequently mixing the cultured cells obtained,their crude extracts, or purified DNA polymerase-constituting proteins.When mixing the two kinds of DNA polymerase-constituting proteins,special procedures are not required, and the DNA polymerase possessingan activity can be obtained simply by mixing the extracts from therespective transformants or the two proteins purified therefrom inappropriate amounts.

The DNA polymerase of the present invention thus obtained provides twobands at positions corresponding to molecular weights of about 90,000 Daand about 140,000 Da on the SDS-PAGE, and these two bands correspondingto the first and second DNA polymerase-constituting proteins,respectively.

As shown in FIG. 3, the DNA polymerase of the present invention exhibitsthe optimum pH is in the neighborhood of 6.5 to 7.0 at 75° C. in apotassium phosphate buffer. When an enzyme activity of the above DNApolymerase is assayed at various temperatures, the enzyme exhibits ahigh activity at 75° to 80° C. However, because the double strandedstructure of the activated DNA used as a substrate for activity assay isdestructed at higher temperatures, an accurate optimum temperature forthe activity of the above enzyme has not been assayed. The above DNApolymerase possesses a high heat stability, retaining not less than 80%of the remaining activity even after a heat treatment at 80° C. for 30minutes, as shown in FIG. 4. This level of the heat stability allows theuse of the above enzyme for PCR method. Also, when assessing theinfluence of aphidicolin, a specific inhibitor of α-type DNApolymerases, it is demonstrated that the activity of the above DNApolymerase is not inhibited even in the presence of 2 mM aphidicolin.

As a result of analyzing the biochemical properties of the purified DNApolymerase, the DNA polymerase of the present invention possesses veryexcellent primer extension activity in vitro. As shown in Table 1, inthe case where DNA polymerase activity is assayed using a substrate in aform resulting from primer annealing to a single stranded DNA (theM13-HT Primer), higher nucleotide incorporating activity as compared tothat of the activated DNA used for usual activity assaying (DNaseI-treated calf thymus DNA) can be demonstrated. When the primerextension ability of the DNA polymerase of the present invention iscompared with that of other DNA polymerases using the above M13-HTPrimer substrate, the DNA polymerase of the present invention exhibitssuperior extension activity as compared to known DNA polymerases derivedfrom Pyrococcus furiousus (Pfu DNA polymerase, manufactured byStratagene) and Taq DNA polymerase derived from Thermus aquaticus(TaKaRa Taq, manufactured by Takara Shuzo Co., Ltd.). Furthermore, whenan activated DNA is added to this reaction system as a competitorsubstrate, the primer extension activities of the above two kinds of DNApolymerases are strongly inhibited, while that of the DNA polymerase ofthe present invention is inhibited at a low level, demonstrating thatthe DNA polymerase of the present invention possesses a high affinityfor substrates of the primer extension type (FIG. 6).

TABLE 1 Relative Activity DNA Polymerase of the Present Pfu DNA Taq DNASubstrates Invention Polymerase Polymerase activated DNA 100 100 100thermal-denatured DNA 340 87 130 M13-HT primer 170 23 90 M13-RNA primer52 0.49 38 poly dA-Oligo dT 94 390 290 poly A-Oligo dT 0.085 — 0.063

Also, the DNA polymerase of the present invention shows excellentperformance when used for the PCR method. In the DNA polymerase derivedfrom Thermus aquaticus, commonly used for the PCR method, it isdifficult to amplify a DNA fragment of not less than 10 kbp using, theabove DNA polymerase alone, and a DNA fragment of not less than 20 kbpcan be amplified when used in combination with another DNA polymerase[Proceedings of the National Academy of Sciences of the USA, 91,2216-2220 (1994)]. Also, the strand length of DNA amplifiable using thePfu DNA polymerase is reportedly at most about 3 kbp. By contrast, whenusing the DNA polymerase of the present invention, the amplification ofa DNA fragment of 20 kbp in length is made possible even when used alonewithout addition of any other enzymes.

Moreover, the DNA polymerase of the present invention which also hasassociated 3′→51′ exonuclease activity is comparable to the Pfu DNApolymerase, known to ensure very high accuracy in DNA synthesis, owingto its high activity in terms of the ratio of the exonuclease activityto the DNA polymerase activity (FIG. 5). Also, the error rate during theDNA synthesis reaction is lower for the DNA polymerase of the presentinvention than that of the Taq DNA polymerase. The various propertiesdemonstrate that the DNA polymerase of the present invention serves veryexcellently as a reagent for genetic engineering techniques such as thePCR method.

The finding of the novel DNA polymerase genes according to the presentinvention also provides an interesting suggestion as follows. In orderto determine the manner in which the region containing the genes forORF3 and ORF4 encoding a novel DNA polymerase is intracellularlytranscribed, the present inventors have analyzed an RNA fractionprepared from Pyrococcus furiousus cells by northern blotting method,RT-PCR method and primer extension method. As a result, it is confirmedthat ORF1 to ORF6 are transcribed from immediately upstream of ORF1 as asingle messenger RNA (mRNA). From the above finding, there is anexpectation that the production of the ORF1 and the ORF2 in cells issubjected to the same control as that for the ORF3 and the ORF4. Whenconsidering in combination with the sequence homologies of ORF1, ORF2,ORF5, and ORF6 to those of CDC6 and CDC18, the CDC6 and the CDC18 beinginvolved in the regulation for initiation of the DNA replication inyeasts, the above expectation suggests that the novel DNA polymerase ofthe present invention is highly likely to be a DNA polymerase importantfor the DNA replication. Since it is also expected that the DNAreplication system of archaebacteria, to which group Pyrococcusfuriousus belongs, is closely related to that of eukaryotic cells, thereis a possibility of the presence of an enzyme similar to the DNApolymerase of the present invention as a DNA. polymerase important forreplication that has not been found in eukaryotes.

It is also expected that thermostable DNA polymerases similar to the DNApolymerase of the present invention are produced in other bacteriabelonging to hyperthermophilic archaebacteria like Pyrococcus furiousus,including, for instance, bacteria other than Pyrococcus furioususbelonging to the genus Pyrococcus; bacteria belonging to the genusPyrodictium; the genus Thermococcus, the genus Staphylothermus, andother genera. When these enzymes are constituted by two DNApolymerase-constituting proteins, like the DNA polymerase of the presentinvention, it is expected that a similar DNA polymerase activity isexhibited by combining one of the two DNA polymerase-constitutingproteins and the DNA polymerase-constituting protein of the presentinvention corresponding to the other DNA polymerase-constitutingprotein.

The thermostable DNA polymerases similar to the DNA polymerase of thepresent invention, produced by the above hyperthermophilicarchaebacteria, are expected to have homology to the DNA polymerase ofthe present invention in terms of its amino acid sequence and the basesequence of the gene encoding thereof. It is therefore possible toobtain the gene for a thermostable DNA polymerase similar to the DNApolymerase of the present invention of which the base sequence is notidentical to that of the DNA polymerase of the present invention butpossesses similar enzyme activities by a process comprising introducinginto an appropriate microorganism a DNA fragment obtained from one ofthe above thermophilic archaebacteria by hybridization using, as aprobe, a gene isolated by the present invention or a portion of theabove base sequence, and assaying the DNA polymerase activity in aheat-treated lysate prepared in the same manner as the above cosmidprotein library by an appropriate method.

The above hybridization can be carried out under the followingconditions. Specifically, a DNA-immobilized membrane is incubated with aprobe at 50° C. for 12 to 20 hours in 6×SSC, wherein 1×SSC indicates0.15 M NaCl, 0.015 M sodium citrate, pH 7.0, containing 0.5% SDS, 0.1%bovine serum albumin, 0.1% polyvinyl pyrrolidone, 0.1% Ficol 400, and0.01% denatured salmon sperm DNA. After termination of the incubation,the membrane is washed, initiating at 37° C. in 2×SSC containing 0.5%SDS, and changing the SSC concentration to 0.1×SSC from the startinglevel, while varying the SSC temperature to 50° C. until the signal fromthe immobilized DNA becomes distinguishable from the background.

Thus, it is possible to obtain a gene for a thermostable DNA polymerasesimilar to the DNA polymerase of the present invention of which the DNApolymerase activity is not identical but of the same level as that ofthe DNA polymerase of the present invention, by introducing into anappropriate microorganism a DNA fragment obtained by a geneamplification reaction using, as a primer, a gene isolated by thepresent invention or a portion of the base sequence of the gene, with aDNA obtained from one of the above thermophilic archaebacteria as atemplate, or a DNA fragment resulting from the thermophilicarchaebacterium by hybridization with the fragment obtained by a geneamplification reaction as a probe, and assaying the DNA polymeraseactivity in the same manner as above.

The present invention is hereinafter described by means of the followingexamples, but the scope of the present invention is not limited only tothose examples. The % values shown in Examples below mean % by weight.

EXAMPLE 1

(1) Preparation of Pyrococcus furiosus Genomic DNA

Pyrococcus furiousus DSM3638 was cultured in the following manner:

A medium having a composition comprising 1% trypton, 0.5% yeast extract,1% soluble starch, 3.5% Jamarin S Solid (Jamarin Laboratory), 0.5%Jamarin S Liquid (Jamarin Laboratory), 0.003% MgSO₄, 0.001% NaCl,0.0001% FeSO₄.7H₂O, 0.0001% CoSO₄, 0.0001% CaCl₂.7H₂O, 0.0001% ZnSO₄,0.1 ppm CuSO₄.5H₂O, 0.1 ppm KAl(SO₄)₂, 0.1 ppm H₃BO₃, 0.1 ppmNa₂MoO₄.2H₂O, and 0.25 ppm NiCl₂.6H₂O was placed in a two-liter mediumbottle and sterilized at 120° C. for 20 minutes. After removal ofdissolved oxygen by sparging with nitrogen gas thereinto, the abovestrain was inoculated into the resulting medium. Thereafter, the mediumwas cultured by kept standing at 95° C. for 16 hours. After terminationof the cultivation, cells were harvested by centrifugation.

The harvested cells were then suspended in 4 ml of 0.05 M Tris-HCl (pH8.0) containing 25% sucrose. To this suspension, 0.8 ml of lysozyme [5mg/ml, 0.25 M Tris-HCl (pH 8.0)] and 2 ml of 0.2 M EDTA were added andincubated at 20° C. for 1 hour. After adding 24 ml of an SET solution[150 mM NaCl, 1 mM EDTA, and 20 mM Tris-HCl (pH 8.0)], 4 ml of 5% SDSand 400 μl of proteinase K (10 mg/ml) were added to the resultingmixture. Thereafter, the resulting mixture was reacted at 37° C. for 1hour. After termination of the reaction, phenol-chloroform extractionand subsequent ethanol precipitation were carried out to prepare about3.2 mg of genomic DNA.

(2) Preparation of Cosmid Protein Library

Four hundred micrograms of the genomic DNA from Pyrococcus furioususDSM3638 was partially digested with Sau3A1 and fractionated by size into35 to 50 kb fractions by density gradient ultracentrifugation method.One microgram of the triple helix cosmid vector (manufactured byStratagene) was digested with XbaI, dephosphorylated using an alkalinephosphatase (manufactured by Takara Shuzo Co., Ltd.), and furtherdigested with BamHI. The resulting treated vector was subjected toligation after mixing with 140 μg of the above 35 to 50 kb DNAfractions. The genomic DNA fragment from Pyrococcus furiousus waspackaged into lambda phage particles by in vitro packaging method using“GIGAPACK GOLD” (manufactured by Stratagene), to prepare a library. Aportion of the obtained library was then transduced into E. coliDH5αMCR. Several transformants out of the resulting transformants wereselected to prepare a cosmid DNA. After confirmation of the presence ofan insert of appropriate size, about 500 transformants were againselected from the above library, and each was separately cultured in 150ml of an LB medium (10 g/l trypton, 5 g/l yeast extract, 5 g/l NaCl, pH7.2) containing 100 μg/ml ampicillin. The resulting culture wascentrifuged, and the harvested cells were suspended in 1 ml of 20 mMTris-HCl at a pH of 8.0, and the resulting suspension was thenheat-treated at 100° C. for 10 minutes. Next, ultrasonication wascarried out, and a heat treatment was carried out again at 100° C. for10 minutes. The lysate obtained as a supernatant after centrifugationwas used as a cosmid protein library.

(3) Assay of DNA Polymerase Activity

The DNA polymerase activity was assayed using calf thymus DNA(manufactured by Worthington) activated by DNase I treatment (activatedDNA) as a substrate. DNA activation and assay of DNA polymerase activitywere carried out by the method described in DNA Polymerase fromEscherichia coli, 263-276 (authored by C. C. Richardson), published byHarper & Row, edited by D. R. Davis.

An assay of enzyme activity was carried out by the following method.Specifically, 50 μl of a reaction solution [20 mM Tris-HCl (pH 7.7), 15mM MgCl₂, 2 mM 2-mercaptoethanol, 0.2 mg/ml activated DNA, 40 μM each ofdATP, dCTP, dGTP and dTTP, 60 nM [³H]-dTTP (manufactured by Amersham)],containing a sample for assaying its activity, was prepared and reactedat 75° C. for 15 minutes. A 40 μl portion of this reaction mixture wasthen spotted onto a DE paper (manufactured by Whatman) and washed with5% Na₂HPO₄ five times. The remaining radioactivity on the DE paper wasassayed using a liquid scintillation counter. The amount of enzyme whichincorporated 10 nmol of [³H]-dTMP per 30 minutes into the substrate DNA,assayed by the above-described enzyme activity assay method, was definedas one unit of the enzyme.

(4) Selection of Cosmid Clones Containing DNA Polymerase Gene

A reaction mixture comprising 20 mM Tris-HCl (pH 7.7), 2 mM MgCl₂, 2 mM2-mercaptoethanol, 0.2 mg/ml activated DNA, 40 μM each of dATP, dCTP,dGTP and dTTP, 60 nM [³H]-dTTP (manufactured by Amersham) was prepared.One μl of 5 clones each of the respective extracts from the cosmidprotein library, namely 5 μl of extracts as for one reaction, was addedto 45 μl of this mixture. After the mixture was reacted at 75° C. for 15minutes, a 40 μl portion of each reaction mixture was spotted onto a DEpaper and washed with 5% Na₂HPO₄ five times. The remaining radioactivityon the DE paper was assayed using a liquid scintillation counter. Agroup found to have some activities by primary assay, wherein one groupconsisted of 5 clones, was separated into one clone each from the 5clones, and then secondary assay was carried out for each clone. Sinceit had been already known that the cosmid DNA library included clonescontaining a known DNA polymerase gene by a hybridization test with thegene as a probe, designated as Clone Nos. 57, 154, 162, and 363, 5clones possessing DNA synthesis activity other than those clones werefound as Clone Nos. 41, 153, 264, 462, and 491.

(5) Preparation of Restriction Endonuclease Map

Cosmids were isolated from the above 5 clones, and each cosmid wasdigested with BamHI. When examining the resulting migration patterns,there were demonstrated several mutually common bands, predicting thatthose 5 clones recombine regions with overlaps and slight shifts. Withthis finding in mind, the DNA inserts in Clone Nos. 264 and 491 weretreated to prepare the restriction endonuclease map. The cosmidsprepared from both clones were digested with various restrictionendonucleases. As a result of determination for respective cleavagesites of KpnI, NotI, PstI, SmaI, XbaI, and XhoI (all manufactured byTakara Shuzo Co., Ltd.), digested into fragments of appropriate sizes, amap as shown in FIG. 1 was obtained.

(6) Subcloning of DNA Polymerase Gene

On the basis of the restriction endonuclease map as shown in FIG. 1,various DNA fragments of about 10 kbp in length were cut out from thecosmid derived from clone No. 264 or 491. The fragments were thensubcloned into the pTV118N or pTV119N vector (manufactured by TakaraShuzo Co., Ltd.). The resulting transformant with each of therecombinant plasmids was then subjected to assaying of the thermostableDNA polymerase activity, to demonstrate that a gene for production of ahighly thermostable DNA polymerase was present an XbaI-XbaI fragment ofabout 10 kbp. A plasmid resulting from recombination of the XbaI-XbaIfragment in the pTV118N vector was then named as plasmid pFU1001, andthe Escherichia coli JM109 transformed with the plasmid was named asEscherichia coli JM109/pFUFU1001.

EXAMPLE 2

Determination of Base Sequence of DNA Fragment Containing Novel DNAPolymerase Gene

The above XbaI-XbaI fragment, containing the DNA polymerase gene, wasagain cut out from the plasmid pFU1001 obtained in Example 1 with XbaI,and blunt-ended using a DNA blunting kit (manufactured by Takara ShuzoCo., Ltd.). The resultant was then ligated to the new pTV118N vector,previously linearized with SmaI, in different orientations to yieldplasmids for preparing deletion mutants. The resulting plasmids werenamed as pFU1002 and pFU1003, respectively. Deletion mutants weresequentially prepared from both ends of the DNA insert using theseplasmids. The Kilo-Sequence deletion kit (manufactured by Takara ShuzoCo., Ltd.) applying Henikoff's method (Gene, 28, 351-359) was used forthe above preparation. The 3′-overhanging type and 5′-overhanging typerestriction endonucleases used were PstI and XbaI, respectively. Thebase sequence of the insert was determined by the dideoxy method usingthe BcaBEST dideoxy sequencing kit (manufactured by Takara Shuzo Co.,Ltd.) with the various deletion mutants as templates.

An 8,450 bp sequence in the base sequence determined is shown by SEQ IDNO:5 in Sequence Listing. As a result of analysis of the base sequence,there were revealed six open reading frames (ORFs) capable of encodingproteins, present at positions corresponding to Base Nos. 123-614(ORF1), 611-1381 (ORF2), 1384-3222 (ORF3), 3225-7013 (ORF4), 7068-7697(ORF5), and 7711-8385 (ORF6) in the base sequence as shown by SEQ IDNO:5 in Sequence Listing. The restriction endonuclease map of the about10 kbp XbaI-XbaI DNA fragment recombined in the plasmid pFU1001 and thelocation of the above-mentioned ORFs thereon are shown in FIG. 2.

In addition, the thermostable DNA polymerase activity was assayed usingthe above various deletion mutants. The results demonstrated that theDNA polymerase activity is lost when the deletion involves the ORF3 andORF4 regions, regardless of whether the deletion started from upstreamor downstream. This finding demonstrated that the translation productsof the ORF3 and the ORF4 were important in the exhibition of the DNApolymerase activity. The base sequence of the ORF3 is shown by SEQ IDNo:1 in Sequence Listing, and the amino acid sequence of the translationproduct of the ORF3 as deduced from the base sequence is SEQ ID NO:2 inSequence Listing, respectively. Also, the base sequence of ORF4 is shownby SEQ ID NO:3 in Sequence Listing, and the amino acid sequence of thetranslation product of ORF4 as deduced from the base sequence is SEQ IDNO:4 in Sequence Listing, respectively.

EXAMPLE 3

Preparation of Purified DNA Polymerase Standard Preparation

The Escherichia coli JM109/pFU1001 obtained in Example 1 was cultured in500 ml of an LB medium (10 g/l trypton, 5 g/l yeast extract, 5 g/l NaCl,pH 7.2) containing ampicillin at a concentration of 100 μg/ml. When theculture broth turbidity reached 0.6 in A₆₀₀, an inducer,isopropyl-β-D-thiogalactoside (IPTG) was added and cultured for 16hours. After harvesting, the harvested cells were suspended in 37 ml ofa sonication buffer [50 mM Tris-HCl, pH 8.0, 0.2 mM 2-mercaptoethanol,10% glycerol, 2.4 mM PMSF (phenylmethanesulfonyl fluoride)] and appliedto an ultrasonic disrupter. Forty-two milliliters of a crude extract wasrecovered as a supernatant by centrifugation at 12,000 rpm for 10minutes, which was then heat-treated at 80° C. for 15 minutes.Centrifugation was again carried out at 12,000 rpm for 10 minutes toyield 33 ml of a heat-treated enzyme solution. The above solution wasthen dialyzed with 800 ml of buffer A [50 mM potassium phosphate, pH6.5, 2 mM 2-mercaptoethanol, 10% glycerol] as an external dialysisliquid for 2 hours×4. After dialysis, 32 ml of the enzyme solution wasapplied to a RESOURCE Q column (manufactured by Pharmacia) which waspreviously equilibrated with buffer A, and subjected to chromatographyusing an FPLC system (manufactured by Pharmacia). A development ofchromatogram was carried out on a linear concentration gradient from 0to 500 mM NaCl. A fraction having a DNA polymerase activity was elutedat 340 mM NaCl.

Ten milliliters of an enzyme solution obtained by collecting as anactive fraction was desalted and concentrated by ultrafiltration, anddissolved in buffer A+150 mM NaCl to yield 3.5 ml of an enzyme solution.The resulting enzyme solution was then applied to a Hi Trap Heparincolumn (manufactured by Pharmacia), previously equilibrated with thesame buffer. A chromatogram was developed on a linear concentrationgradient from 150 to 650 mM NaCl using an FPLC system, to yield anactive fraction eluted at 400 mM NaCl. Five milliliters of this fractionwas concentrated to 120 μl of a solution including 50 mM potassiumphosphate, pH 6.5, 2 mM 2-mercaptoethanol, and 75 mM NaCl by repeatingdesalting and concentration using ultrafiltration. The resultingconcentrated solution was then applied to a gel filtration column ofSuperose 6 (manufactured by Pharmacia), previously equilibrated with thesame buffer, and eluted with the same buffer. As a result, a fractionhaving a DNA polymerase activity was eluted at positions correspondingto retention times of 34.7 minutes and 38.3 minutes. It is suggestedfrom the results of comparison with the elution position of molecularweight markers under the same conditions that these activity peaks havemolecular weights of about 385 kDa and about 220 kDa, respectively.These molecular weights corresponded to a complex formed by thetranslation product of ORF3 and the translation product of ORF4 in amolar ratio of 1:2 and another complex formed by the above translationproducts in a molar ratio of 1:1, respectively. For the former peak,however, since a possibility that a complex is formed by the twotranslation products in a 2:2 molar ratio cannot be negated, themolecular weight determination error increases as the molecular weightincreases.

EXAMPLE 4

(1) Biochemical Properties of DNA Polymerase

For a DNA polymerase preparation forming a complex of the translationproducts of ORF3 and ORF4 obtained in Example 3, namely the first DNApolymerase-constituting protein and the second DNApolymerase-constituting protein in a ratio at 1:1, optimum MgCl₂ and KClconcentrations were firstly assayed. The DNA polymerase activity wasassayed in a reaction system containing 20 mM Tris-HCl, pH 7.7, 2 mM2-mercaptoethanol, 0.2 mg/ml activated DNA, and 40 μM each of dATP,dGTP, dCTP and dTTP in the presence of 2 mM MgCl₂, while the KClconcentration was step by step increased from 0 to 200 mM KCl for each20 mM increment. As a result, the maximum activity was exhibited at aKCl concentration of 60 mM. Next, the DNA polymerase activity wasassayed in the same reaction system but in the presence of 60 mM KCl inthis time, while the MgCl₂ concentration was step by step increased from0.5 to 25 mM MgCl₂ for each 2.5 mM increment, to compare at eachconcentration. In this case, the maximum activity was exhibited at anMgCl₂ concentration of 10 mM, and alternatively, in the absence of KClthe maximum activity was exhibited at an MgCl₂ concentration of 17.5 mM.

The optimum pH was then assayed. The DNA polymerase activity was assayedat 75° C. by using potassium phosphate buffers at various pH levels, andpreparing a reaction mixture comprising 20 mM potassium phosphate, 15 mMMgCl₂, 2 mM 2-mercaptoethanol, 0.2 mg/ml activated DNA, 40 μM each ofdATP, dCTP, dGTP and dTTP, and 60 nM [³H]-dTTP. The results are shown inFIG. 3, wherein the abscissa indicates the pH, and the ordinateindicates the radioactivity incorporated in high-molecular DNA. As shownin the figure, the DNA polymerase of the present invention exhibited themaximum activity at a pH of 6.5 to 7.0. When Tris-HCl was used in placeof potassium phosphate, the activity increased with alkalinity, and themaximum activity was exhibited at a pH of 8.02, the highest pH levelused in the assay.

The heat stability of the DNA polymerase of the present invention wasassayed as follows: The purified DNA polymerase was prepared to yield amixture containing 20 mM Tris-HCl (pH 7.7), 2 mM 2-mercaptoethanol, 10%glycerol, and 0.1% bovine serum albumin, and the resulting mixture wasincubated at various temperatures for 30 minutes. The remaining DNApolymerase activity was assayed. The results are shown in FIG. 4,wherein the abscissa indicates the incubation temperature, and theordinate indicates the remaining activity. As shown in the figure, thepresent enzyme retained not less than 80% remaining activity even afterheat treatment at 80° C. for 30 minutes.

In order to compare the modes of inhibition by inhibitors, the modes ofinhibition of the DNA polymerase of the present invention and an α-typeDNA polymerase derived from Pyrococcus furiousus (Pfu DNA polymerase,manufactured by Stratagene), a known DNA polymerase, were compared usinga specific inhibitor of α-type DNA polymerases, aphidicolin. Theactivity changes were examined, while the aphidicolin concentration wasincreased from 0 to 2.0 mM in the presence of 20 mM Tris-HCl, pH 7.7, 15mM MgCl₂, 2 mM 2-mercaptoethanol, 0.2 mg/ml activated DNA, and 40 μMeach of dATP, dGTP, dCTP and dTTP. As a result, the activity of the PfuDNA polymerase was decreased to 20% of the original activity at 1.0 mM,while the novel DNA polymerase of the present invention was notinhibited at all even at 2.0 mM.

(2) Primer Extension Reaction

Next, in order to compare the selectivity of the DNA polymerase of thepresent invention for different forms of substrate DNA, the followingtemplate-primer was examined. Aside from the activated DNA used forconventional assay of the activity, those prepared as substrates includea thermal-denatured DNA prepared by treating the activated DNA at 85° C.for 5 minutes; M13-HT Primer prepared by annealing a 45-base syntheticdeoxyribooligonucleotide of the sequence as shown by SEQ ID NO:6 inSequence Listing as a primer to the M13 phage single stranded DNA(M13mp18 ssDNA, manufactured by Takara Shuzo Co., Ltd.); M13-RNA Primerprepared by annealing a 17-base synthetic ribooligonucleotide of thesequence as shown by SEQ ID NO:7 in Sequence Listing as a primer to thesame M13 phage single stranded DNA; Poly dA-Oligo dT prepared by mixingpolydeoxyadenosine (Poly dA, manufactured by Pharmacia) andoligodeoxythymidine (Oligo dT, manufactured by Pharmacia) in a 20:1molar ratio; and Poly A-Oligo dT prepared by mixing polyadenosine (PolyA, manufactured by Pharmacia) and oligodeoxythymidine in a 20:1 molarratio.

The DNA polymerase activity was assayed using these substrates in placeof the activated DNA. The relative activity of each substrate when theactivity obtained in the case of using an activated DNA as a substrateis defined as 100 is shown in Table 1. For comparison, the Pfu DNApolymerase and the Taq DNA polymerase derived from Thermus aquaticus(TaKaRa Taq, manufactured by Takara Shuzo Co., Ltd.) were also examinedin the same manner. As shown in Table 1, in comparison with other DNApolymerases, the novel DNA polymerase of the present invention exhibitedhigher activity when the substrate used was the M13-HT Primer ratherthan the activated DNA, demonstrating that the novel DNA polymerase ofthe present invention is especially suitable for primer extensionreaction.

The primer extension activity was further investigated extensively. TheM13-HT Primer, previously labeled with [γ-³²P]-ATP (manufactured byAmersham) and T4 polynucleotide kinase (manufactured by Takara ShuzoCo., Ltd.) at the 5′-end, was used as a substrate. Ten microliters of areaction mixture [20 mM Tris-HCl (pH 7.7), 15 mM MgCl₂, 2 mM2-mercaptoethanol, 270 μM each of dATP, dGTP, dCTP and dTTP] containingthe above substrate in a final concentration of 0.05 μg/pl and variousDNA polymerases in amounts providing 0.05 units of activity as assayedwith the activated DNA as a substrate was reacted at 75° C. for 1, 2, 3,or 4 minutes. After termination of the reaction, 2 μl of a reaction stopsolution (95% formaldehyde, 20 mM EDTA, 0.05% bromophenol blue, 0.05%xylenecyanol) was added, subjected to thermal denaturation treatment at95° C. for 3 minutes. Two microliters of the reaction mixture was thensubjected to electrophoresis using polyacrylamide gel containing 8 Murea and subsequently subjected to a preparation of autoradiogram. Also,in order to examine the extension activity in the presence of theactivated DNA as a competitor substrate, the activated DNA was added tothe above reaction mixture to a final concentration of 0.4 μg/ml, andsubjected to a preparation of an autoradiogram by the same procedures asdescribed above. The autoradiogram obtained is shown in FIG. 6.

In the figure, Pol, Pfu, and Taq show the results for the DNA polymeraseof the present invention, the Pfu DNA polymerase and the Taq DNApolymerase, respectively. In addition, 1, 2, 3, and 4 each indicatesreaction time (min). In the figure, the representation “−” and “+” showthe results obtained in the absence and in the presence, respectively,of the activated DNA. The lanes G, A, T, and C at the left end of thefigure also show the results of electrophoresis of the reaction productsobtained by a chain termination reaction by the dideoxy method using thesame substrate as above, which were used to estimate the length of eachextension product. As shown in the figure, the DNA polymerase of thepresent invention exhibited superior primer extension activity thanthose of the Pfu DNA polymerase and the Taq DNA polymerase. It was alsoshown that the DNA polymerase of the present invention was unlikely tobe inhibited by the activated DNA, in contrast to the Taq DNApolymerase, which exhibited relatively higher primer extension activityin the absence of the activated DNA, was markedly inhibited by theaddition of the activated DNA in great excess. From the above finding,it was confirmed that the DNA polymerase of the present inventionpossesses high affinity especially to primer extension type substrateshaving a form in which a single primer was annealed to a single strandedtemplate DNA.

(3) Presence or Absence of Associated Exonuclease Activity

The exonuclease activity of the DNA polymerase of the present inventionwas assessed as follows: As a substrate for 5′→3′ exonuclease activitydetection, a DNA fragment labeled with ³²P at the 5′-end was prepared bya process comprising digesting a pUC119 vector (manufactured by TakaraShuzo Co., Ltd.) with SspI (manufactured by Takara Shuzo Co., Ltd.),separating the resulting 386 bp DNA fragment by agarose gelelectrophoresis, purifying the fragment, and labeling with [γ-³²P]-ATPand polynucleotide kinase. Also, as a substrate for 3′→5′ exonucleaseactivity detection, a DNA fragment labeled with ³²P at 3′-end wasprepared by a process comprising digesting a pUC119 vector with Sau3AI,separating the resulting 341 bp DNA fragment by agarose gelelectrophoresis, purifying the fragment, and carrying out a fill-inreaction using [γ-³²P]-CTP (manufactured by Amersham) and the Klenowfragment (manufactured by Takara Shuzo Co., Ltd.). The labeled DNAs werepurified by gel filtration with NlCK COLUMN (manufactured by Pharmacia)and used in the subsequent reaction. To a reaction solution [20 mMTris-HCl (pH 7.7), 15 mM MgCl₂, 2 mM 2-mercaptoethanol] containing 1 ngof these labeled DNAS, 0.015 units of DNA polymerase was added, and theresulting mixture was reacted at 75° C. for 2.5, 5, and 7.5 minutes. TheDNAs were precipitated by adding ethanol. The radioactivity existing inthe supernatant was assayed using a liquid scintillation counter, andthe amount of degradation by the exonuclease activity was calculated.The DNA polymerase of the present invention was shown to possess potent3′→5′ exonuclease activity, while no 5′→3′exonuclease activity wasobserved. The 3′→5′ exonuclease activity of the Pfu DNA polymerase,known to possess potent 3′→5′ exonuclease activity, was also assayed inthe same manner as above. The results are together shown in FIG. 5.

In the figure, the abscissa indicates the reaction time, and theordinate indicates the ratio of radioactivity released into thesupernatant relative to the radioactivity contained in the entirereaction mixture. Also, the open circles indicate the results for theDNA polymerase of the present invention, and the solid circles indicatethose for the Pfu DNA polymerase. As shown in the figure, the DNApolymerase of the present invention showed potent 3′→5′ exonucleaseactivity of the same level as that of the Pfu DNA polymerase, known topossess high accuracy of DNA synthesis owing to high 3′→5′ exonucleaseactivity.

(4) Comparison of Accuracy of DNA Synthesis Reaction

The accuracy of DNA synthesis reaction by DNA polymerases was examinedusing a pUC118 vector (manufactured by Takara Shuzo Co., Ltd.),partially made single stranded (gapped duplex plasmid, as a template.The single stranded pUC118 vector was prepared by the method describedin Molecular Cloning: A Laboratory Manual, 2nd ed., 4.44-4.48, publishedby Cold Spring Harbor Laboratory in 1989, edited by T. Maniatis et al.,using a helper phage M13KO7 (manufactured by Takara Shuzo Co., Ltd.)with Escherichia coli MV1184 (manufactured by Takara Shuzo Co., Ltd.) asa host. The double stranded DNA was prepared by digesting the pUC118vector with PvuII (manufactured by Takara Shuzo Co., Ltd.), subjectingthe digested vector to agarose gel electrophoresis, and recovering a DNAfragment of about 2.8 kbp.

One microgram of the above single stranded DNA and 2 μg of the doublestranded DNA were mixed to make 180 μl of a mixture with steriledistilled water, and the solution was then incubated at 70° C. for 10minutes. Thereafter, twenty microliters of 20×SSC was added to theresulting mixture, and the mixture was further kept standing at 60° C.for 10 minutes. The DNA was recovered by subjecting to ethanolprecipitation. A portion thereof was subjected to agarose gelelectrophoresis, and it was confirmed that a gapped duplex plasmid wasobtained. Thirty microliters of a reaction mixture [10 mM Tris-HCl, pH8.5, 50 mM KCl, 10 mM Mgcl₂, 1 mM each of dATP, dCTP, dGTP and dTTP],containing an amount one-tenth that of the resulting gapped duplexplasmid was incubated at 700° C. for 3 minutes, after which 0.5 units ofDNA polymerase was added thereto, and a DNA synthesis reaction wascarried out at 70° C. for 10 minutes. After termination of the reaction,Escherichia coli DH5α (manufactured by BRL) was transformed using 10 μlof the reaction mixture. The resulting transformant was cultured at 37°C. for 18 hours on an LB plate containing 100 μg/ml ampicillin, 0.1 mMIPTG, and 40 μg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside. The whiteor blue colonies formed on the plate were counted, and the formationrate of white colonies which were resulted from a DNA synthesis errorwas calculated. As a result, the white colony formation rate (%) was3.18% when the Tag DNA polymerase was used as the DNA polymerase, incontrast to a lower formation rate of 1.61% when the DNA polymerase ofthe present invention was used.

(5) Application to PCR

In order to compare the performance of the DNA polymerase of the presentinvention in PCR with that of the Taq DNA polymerase, PCR was carriedout with λ-DNA as a template. The reaction mixture for the DNApolymerase of the present invention had the following composition: 10 mMTris-HCl (pH 9.2), 3.5 mM MgCl₂, 75 mM KCl, 400 μM each of dATP, dCTP,dGTP and dTTP, 0.01% bovine serum albumin (BSA), and 0.1% Triton X-100.The reaction solution for the Taq DNA polymerase had the followingcomposition: 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl, and 400μM each of dATP, dCTP, dGTP and dTTP. Fifty microliters of a reactionmixture containing 5.0 ng/50 μl λ-DNA (manufactured by Takara Shuzo Co.,Ltd.), 10 pmol/50 μl each of primer λ1 and primer λ11, and 3.7 units/50μl DNA polymerase was prepared. The base sequences of the primer λ1 andthe primer λ11 are shown by SEQ ID NO:8 and SEQ ID NO:9 in SequenceListing, respectively. After, a 30-cycle PCR was carried out with theabove reaction mixture, wherein one cycle is defined at 98° C. for 10seconds and at 68° C. for 10 seconds. Five microliters of the reactionmixture was subjected to agarose gel electrophoresis, and the amplifiedDNA fragment was confirmed by staining with ethidium bromide. As aresult, it was demonstrated that the DNA fragment amplification was notfound when the Taq DNA polymerase was used, in contrast to the DNApolymerase of the present invention where amplification of a DNAfragment of about 20 kbp was confirmed.

The experiment was then carried out by changing the primer to the primerλ1 and the primer λ10. The base sequence of the primer λ10 is shown bySEQ ID NO:10 in Sequence Listing. Twenty-five microliters of a reactionmixture having a similar composition to that shown above and containing2.5 ng of λ-DNA, 10 pmol of the primer λ1 and the primer λ10,respectively, and 3.7 units of DNA polymerase was prepared. The reactionmixture was reacted in 5 cycles under the same reaction conditions asthose described above, and 5 μl of the reaction mixture was subjected toagarose gel electrophoresis and stained with ethidium bromide. It wasdemonstrated that no specific amplification was observed when the TaqDNA polymerase was used, in contrast to the DNA polymerase of thepresent invention where a DNA fragment of about 15 kbp was amplified.

EXAMPLE 5

(1) Construction of Plasmid for Expression of ORF3

Translation Product Alone

PCR was carried out using a mutant plasmid 6-82 as a template, themutant plasmid being prepared by deleting the portion immediatelydownstream of the ORF3 from the DNA insert in the plasmid pFU1002described in Example 2, wherein the ORF1 to the ORF6 were locateddownstream of the lac promoter on the vector and also using a primer M4(manufactured by Takara Shuzo Co., Ltd) and the primer NO:3 whose basesequence is shown by SEQ ID:11 in Sequence Listing. The DNA polymeraseused for the PCR was the Pfu DNA polymerase (manufactured byStratagene), which possessed high accuracy of synthesis reaction. A25-cycle reaction of 100 μl of a reaction mixture for PCR [20 mMTris-HCl, pH 8.2, 10 mM KCl, 20 mM MgCl₂, 6 mM (NH₄)₂SO₄, 0.2 mM each ofdATP, dCTP, dGTP and dTTP, 1% Triton X-100, 0.01% BSA] containing 1 ngof a template DNA, 25 pmol of each primer, and 2.5 units of the Pfu DNApolymerase was carried out, wherein one cycle is defined as at 94° C.for 0.5 minutes, at 55° C. for 0.5 minutes and at 72° C. for 2 minutes.The amplified DNA fragment of about 2 kbp was digested with NcoI andSphI (each manufactured by Takara Shuzo Co., Ltd.) and inserted intobetween the NcoI-SphI sites of the pTV118N vector (manufactured byTakara Shuzo Co., Ltd.) to prepare a plasmid pFU-ORF3. The DNA insert inthe above plasmid contains ORF3 alone in translatable conditions.

(2) Construction of Plasmid for Expression of ORF4 Translation ProductAlone

PCR was carried out using a mutant plasmid 6-2 as a template, the mutantplasmid being prepared by deleting the portion downstream of the centerportion of the ORF4 from the DNA insert in the above-described plasmidpFU1002, the primer M4, and the primer NO4 of which the base sequence isshown by SEQ ID NO:12 in Sequence Listing. The reaction was carried outunder the same conditions as those for Example 5-(1) described above,except that the template DNA was replaced with the plasmid 6-2, and theprimer NO3 was replaced with the primer NO:04. A DNA fragment of about1.6 kbp obtained by digesting the above amplified DNA fragment with NcoIand NheI (manufactured by Takara Shuzo Co., Ltd.), together with anabout 3.3 kbp NheI-Sal fragment, including the latter portion of ORF4,isolated from the above plasmid pFU1002 was inserted between theNcoI-XhoI sites of a pET15b vector (manufactured by Novagen) to preparea plasmid pFU-ORF4. The DNA insert in the plasmid contains ORF4 alone intranslatable conditions.

(3) Reconstitution of DNA Polymerase with ORF3 and ORF4

Translation Products

The Escherichia coli JM109 transformed with the above-described plasmidpFU-ORF3, Escherichia coli JM109/pFU-ORF3, and the Escherichia coliHMS174 transformed with the above-described plasmid pFU-ORF4,Escherichia coli HMS174/pFU-ORF4, were separately cultured, and then thetranslation products of the two ORFs expressed in their cells werepurified. The cultivation of the transformants and the preparation ofthe crude extracts were carried out by the methods described in Example3. Purification of both translation products was carried out usingcolumns such as RESOURCE Q, HiTrap Heparin, and Superose 6, while thebehaviors of the translation products on SDS-PAGE were monitored. It wasconfirmed that although neither of the ORF translation products thuspurified exhibited the DNA polymerase activity when assayed alone,thermostable DNA polymerase activity was exhibited when they were mixedtogether.

Industrial Applicability

The present invention can provide a novel DNA polymerase possessing bothhigh primer extensibility and high 3′→5′ exonuclease activity. Theenzyme is suitable for its use for PCR method, which is useful for areagent for genetic engineering investigation. It is also possible toproduce the enzyme by genetic engineering using the genes encoding theDNA polymerase of the present invention.

What is claimed is:
 1. An isolated and purified DNA polymerase,characterized in that said DNA polymerase possesses the followingproperties: 1) exhibiting higher polymerase activity when assayed byusing as a substrate a complex resulting from primer annealing to asingle stranded template DNA, compared to using an activated DNA as asubstrate; 2) possessing a 3′→5′ exonuclease activity; 3) amplifying aDNA fragment of about 20 kbp, when polymerase chain reaction (PCR) iscarried out using λ-DNA as a template under the following conditions:PCR conditions: (a) a composition of reaction mixture: containing 10 mMTris-HCl (pH 9.2), 3.5 mM MgCl₂, 75 mM KCl, 400 μM each of dATP, dCTP,dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ IDNO:9), and 3.7 units/50 μl DNA polymerase; (b) reaction conditions:carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C.for 10 seconds and at 68° C. for 10 minutes; wherein said DNA polymerasecomprises a first polypeptide and a second polypeptide, which arenon-covalently bonded to form a complex, wherein said first polypeptidecomprises: (I) the amino acid sequence encoded by a DNA of SEQ ID NO:1,or (II) an amino acid sequence encoded by a DNA, the complement thereofhybridizing to the DNA of SEQ ID NO:1 under stringent conditions; andwherein said second polypeptide comprises: (I) the amino acid sequenceencoded by a DNA of SEQ ID NO:3, or (II) an amino acid sequence encodedby a DNA, the complement thereof hybridizing to the DNA of SEQ ID NO:3under stringent conditions.
 2. The isolated and purified DNA polymeraseaccording to claim 1, characterized in that said DNA polymerase exhibitsa lower error rate in DNA synthesis as compared to Taq DNA polymerase.3. The isolated and purified DNA polymerase according to claim 1,wherein the molecular weight as determined by gel filtration method isabout 220 kDa or about 385 kDa.
 4. An isolated and purified DNApolymerase acccording to claim 1, characterized in that said DNApolymerase exhibits a DNA polymerase activity wherein said DNApolymerase comprises a first polypeptide and a second polypeptide, whichare non-covalently bonded to form a complex, wherein the molecularweight of said first polypeptide is about 90,000 Da and the molecularweight of said second polypeptide is about 140,000 Da as determined bySDS-PAGE, and wherein said DNA polymerase possesses the followingproperties: 1) exhibiting higher polymerase activity when assayed byusing as a substrate a complex resulting from primer annealing to asingle stranded template DNA, compared to using an activated DNA as asubstrate; 2) possessing a 3′→5′ exonuclease activity; 3) amplifying aDNA fragment of about 20 kbp, when polymerase chain reaction (PCR) iscarried out using λ-DNA as a template under the following conditions:PCR conditions: (a) a composition of reaction mixture: containing 10 mMTris-HCl (pH 9.2), 3.5 mM MgCl₂, 75 mM KCl, 400 μM each of dATP, dCTP,dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ IDNO:9), and 3.7 units/50 μl DNA polymerase; (b) reaction conditions:carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C.for 10 seconds and at 68° C. for 10 minutes.
 5. An isolated and purifiedfirst polypeptide comprising the amino acid sequence as shown by SEQ IDNO:2, wherein the first polypeptide exhibits DNA polymerase activity andthe following properties: 1) exhibiting higher polymerase activity whenassayed by using as a substrate a complex resulting from primerannealing to a single stranded template DNA, compared to using anactivated DNA as a substrate; 2) possessing a 3′→5′ exonucleaseactivity; 3) amplifying a DNA fragment of about 20 kbp, when polymerasechain reaction (PCR) is carried out using λ-DNA as a template under thefollowing conditions: PCR conditions: (a) a composition of reactionmixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl₂, 75 mM KCl,400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serum albumin,0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ IDNO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNA polymerase; (b)reaction conditions: carrying out a 30-cycle PCR, wherein one cycle isdefined as at 98° C. for 10 seconds and at 68° C. for 10 minutes, whencombined with a second polypeptide of the amino acid sequence as shownby SEQ ID NO:4.
 6. An isolated and purified second polypeptidecomprising the amino acid sequence as shown by SEQ ID NO:4, wherein thesecond polypeptide exhibits DNA polymerase activity and the followingproperties: 1) exhibiting higher polymerase activity when assayed byusing as a substrate a complex resulting from primer annealing to asingle stranded template DNA, compared to using an activated DNA as asubstrate; 2) possessing a 3′→5′ exonuclease activity; 3) amplifying aDNA fragment of about 20 kbp, when polymerase chain reaction (PCR) iscarried out using λ-DNA as a template under the following conditions:PCR conditions: (a) a composition of reaction mixture: containing 10 mMTris-HCl (pH 9.2), 3.5 mM MgCl₂, 75 mM KCl, 400 μM each of dATP, dCTP,dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ IDNO:9), and 3.7 units/50 μl DNA polymerase; (b) reaction conditions:carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C.for 10 seconds and at 68° C. for 10 minutes, when combined with a firstpolypeptide of the amino acid sequence as shown by SEQ ID NO:2.
 7. Anisolated DNA encoding a first polypeptide comprising a base sequenceselected from the group consisting of: (I′) a base sequence encoding theamino acid sequence as shown by SEQ ID NO:2, (II′) the base sequence asshown by SEQ ID NO:1, and (III′) a base sequence of a DNA, thecomplement thereof hybridizing to DNA of SEQ ID NO:1 under stringentconditions, wherein the first polypeptide encoded by the base sequenceexhibits DNA polymerase activity and the following properties: 1)exhibiting higher polymerase activity when assayed by using as asubstrate a complex resulting from primer annealing to a single strandedtemplate DNA, compared to using an activated DNA as a substrate; 2)possessing a 3′→5′ exonuclease activity; 3) amplifying a DNA fragment ofabout 20 kbp, when polymerase chain reaction (PCR) is carried out usingλ-DNA as a template under the following conditions: PCR conditions: (a)a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2),3.5 mM MgCl₂, 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01%bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl. λ-DNA, 10pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7units/50 μl DNA polymerase; (b) reaction conditions: carrying out a30-cycle PCR, wherein one cycle is defined as at 98° C. for 10 secondsand at 68° C. for 10 minutes, when combined with a second polypeptide ofthe amino acid sequence as shown by SEQ ID NO:4.
 8. An isolated DNAencoding a second polypeptide comprising a base sequence selected fromthe group consisting of: (I′) a base sequence encoding the amino acidsequence as shown by SEQ ID NO:4, (II′) the base sequence as shown bySEQ ID NO:3, and (III′) a base sequence of a DNA, the complement thereofhybridizing to DNA of SEQ ID NO:3 under stringent conditions, whereinthe second polypeptide encoded by the base sequence exhibits DNApolymerase activity and the following properties: 1) exhibiting higherpolymerase activity when assayed by using as a substrate a complexresulting from primer annealing to a single stranded template DNA,compared to using an activated DNA as a substrate; 2) possessing a 3′→5′exonuclease activity; 3) amplifying a DNA fragment of about 20 kbp, whenpolymerase chain reaction (PCR) is carried out using λ-DNA as a templateunder the following conditions: PCR conditions: (a) a composition ofreaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl₂, 75mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serumalbumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50 μl primer λ1(SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNApolymerase; (b) reaction conditions: carrying out a 30-cycle PCR,wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C.for 10 minutes, when combined with a first polypeptide of the amino acidsequence as shown by SEQ ID NO:2.
 9. A method for producing a DNApolymerase, comprising the steps of: culturing a transformant containingboth a gene encoding a first polypeptide, which contains DNA encodingthe amino acid sequence of SEQ ID NO:2, a DNA of the base sequence ofSEQ ID NO:1, or a DNA, the complement thereof which hybridizes to thebase sequence of SEQ ID NO:1 under stringent conditions, and a geneencoding a second polypeptide, which contains DNA encoding the aminoacid sequence of SEQ ID NO:4, a DNA of the base sequence of SEQ ID NO:3,or a DNA, the complement thereof which hybridizes to the base sequenceof SEQ ID NO:3 under stringent conditions; and collecting the DNApolymerase from the resulting culture wherein the molecular weight ofsaid first polypeptide is about 90,000 Da and that of said secondpolypeptide is about 140,000 Da as determined by SDS-PAGE, and whereinsaid DNA polymerase possesses the following properties: 1) exhibitinghigher polymerase activity when assayed by using as a substrate acomplex resulting from primer annealing to a single stranded templateDNA, compared to using an activated DNA as a substrate; 2) possessing a3′→5′ exonuclease activity; 3) amplifying a DNA fragment of about 20kbp, when polymerase chain reaction (PCR) is carried out using λ-DNA asa template under the following conditions: PCR conditions: (a) acomposition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5mM MgCl₂, 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01%bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50μl DNA polymerase; (b) reaction conditions: carrying out a 30-cycle PCR,wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C.for 10 minutes.
 10. A method for producing a DNA polymerase, comprisingthe steps of: culturing a transformant containing a gene encoding afirst polypeptide, which contains DNA encoding the amino acid sequenceof SEQ ID NO:2, a DNA of the base sequence of SEQ ID NO:1, or a DNA, thecomplement thereof which hybridizes to the base sequence of SEQ ID NO:1under stringent conditions, and a transformant containing a geneencoding a second polypeptide, which contains DNA encoding the aminoacid sequence of SEQ ID NO:4, a DNA of the base sequence of SEQ ID NO:3,or a DNA, the complement thereof which hybridizes to the base sequenceof SEQ ID NO:3 under stringent conditions; combining the firstpolypeptide contained in the resulting culture and the secondpolypeptide contained in the resulting culture, thereby allowingnon-covalent bonding to form a DNA polymerase as a complex; andcollecting the DNA polymerase, wherein the molecular weight of saidfirst polypeptide is about 90,000 Da and the molecular weight of saidsecond polypeptide is about 140,000 Da as determined by SDS-PAGE, andwherein said DNA polymerase possesses the following properties: 1)exhibiting higher polymerase activity when assayed by using as asubstrate a complex resulting from primer annealing to a single strandedtemplate DNA, compared to using an activated DNA as a substrate; 2)possessing a 3′→5′ exonuclease activity; 3) amplifying a DNA fragment ofabout 20 kbp, when polymerase chain reaction (PCR) is carried out usingλ-DNA as a template under the following conditions: PCR conditions: (a)a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2),3.5 mM MgCl₂, 75 mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01%bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50μl DNA polymerase; (b) reaction conditions: carrying out a 30-cycle PCR,wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C.for 10 minutes.
 11. A recombinant first polypeptide encoded by theisolated DNA of claim 7, wherein the first polypeptide exhibits DNApolymerase activity and the following properties: 1) exhibiting higherpolymerase activity when assayed by using as a substrate a complexresulting from primer annealing to a single stranded template DNA,compared to using an activated DNA as a substrate; 2) possessing a 3′→5′exonuclease activity; 3) amplifying a DNA fragment of about 20 kbp, whenpolymerase chain reaction (PCR) is carried out using λ-DNA as a templateunder the following conditions: PCR conditions: (a) a composition ofreaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl₂, 75mM KCl, 400 μM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serumalbumin, 0.1% Triton X-100, 5.0 ng/50 μl λ-DNA, 10 pmole/50 μl primer λ1(SEQ ID NO:8), primer λ11 (SEQ ID NO:9), and 3.7 units/50 μl DNApolymerase; (b) reaction conditions: carrying out a 30-cycle PCR,wherein one cycle is defined as at 98° C. for 10 seconds and at 68° C.for 10 minutes, when combined with a second polypeptide of the aminoacid sequence as shown by SEQ ID NO:4.
 12. A recombinant secondpolypeptide encoded by the isolated DNA of claim 8, wherein the secondpolypeptide exhibits DNA polymerase activity and the followingproperties: 1) exhibiting higher polymerase activity when assayed byusing as a substrate a complex resulting from primer annealing to asingle stranded template DNA, compared to using an activated DNA as asubstrate; 2) possessing a 3′→5′ exonuclease activity; 3) amplifying aDNA fragment of about 20 kbp, when polymerase chain reaction (PCR) iscarried out using λ-DNA as a template under the following conditions:PCR conditions: (a) a composition of reaction mixture: containing 10 mMTris-HCl (pH 9.2), 3.5 mM MgCl₂, 75 mM KCl, 400 μM each of dATP, dCTP,dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50μl λ-DNA, 10 pmole/50 μl primer λ1 (SEQ ID NO:8), primer λ11 (SEQ IDNO:9), and 3.7 units/50 μl DNA polymerase; (b) reaction conditions:carrying out a 30-cycle PCR, wherein one cycle is defined as at 98° C.for 10 seconds and at 68° C. for 10 minutes, when combined with a firstpolypeptide of the amino acid sequence as shown by SEQ ID NO:2.